Project Summary Breast cancer is the most prevalent cancer among women worldwide. The basal-like subtype of breast cancer is associated with a particularly poor prognosis, yet the molecular mechanisms leading to the origin and pathogenesis of this disease are inadequately understood. Our data has indicated that the Hippo signaling pathway is an important regulator of cell fate and progenitor status in the mammary epithelium. Central to Hippo pathway signaling are the transcriptional regulators YAP and TAZ (YAP/TAZ), which when localized to the nucleus drive transcriptional events that are required for basal stem cell traits. Nuclear YAP/TAZ activity is restricted through the tumor suppressor kinases LATS1/2, which are required to maintain homeostasis in the mammary epithelium. LATS1/2 have been shown to be downregulated in breast cancers, suggesting that dysregulation of these kinases contributes to breast cancer pathology. In this proposal, we aim to use genetic mouse models and ex vivo analysis of primary cells to define how loss of LATS1/2 contributes to the pathogenesis of breast cancer. We have shown that in the homeostatic mammary gland, luminal epithelial cells exhibit active LATS1/2, with restricted YAP/TAZ activity. Our data demonstrate that conditional deletion of LATS1/2 in the luminal mammary epithelium leads to the rapid proliferation of a population of cells within the mammary duct. Notably, expanded cells display traits associated with luminal progenitors, a proposed cell of origin of basal-like and triple-negative breast cancers. Through lineage tracing, our data suggest that the proliferating lesions are comprised of a heterogeneous mixture of LATS1/2-null and LATS1/2-WT cells, and exhibit significant nuclear localization of YAP/TAZ. Furthermore, LATS1/2-null cells derived from our mice are capable of forming tumors when injected subcutaneously into wild-type mice. Our goals in this proposal are to: 1) define how loss of LATS1/2 affects mammary gland homeostasis and contributes to the development of invasive carcinoma through the activity of YAP/TAZ; and 2) identify potential therapeutic targets for LATS1/2- null mammary carcinomas using transcriptional profiling. Collectively, our proposed studies will provide crucial insight into the molecular aberrations underlying the development of breast cancer, which we hope will lead to more impactful treatments and diagnostic strategies for this disease in the future.